RNA interference (RNAi) refers to a phenomenon where a double stranded RNA consisting of a sense RNA having a homologous sequence with mRNA of a target gene and an antisense RNA having a complementary sequence therewith is introduced to cells so as to selectively induce degradation of mRNA of the target gene or to suppress expression of the target gene. RNAi was first found in nematodes, and at present, it is observed in various organisms including yeasts, insects, plants, and humans as a highly preserved biological phenomenon.
Small interference RNA (siRNA), as a material of inducing RNAi, refers to a short RNA double helical strand consisting of about 20 to 30 nucleotides. Introduction of siRNA into cells enables to target mRNA of which the base sequence is complementary to the siRNA, thereby suppressing gene expression. Hence, siRNA has gained interest as an efficient means capable of controlling a life process to be a target by virtue of its therapeutic effects against diseases, easy preparation and high target selectivity.
Currently, cancers, virus infection diseases, autoimmune diseases, and neurodegenerative diseases have been studied as diseases to be treated by use of siRNAs, and their potentials as therapeutic agents for age-related macular degeneration (Bevasiranib; Opko Health, Inc., Miami, Fla., USA; clinical phase III) and respiratory syncytial virus infection (ALN-RSV01; Alnylam, Cambridge, Mass., USA; clinical phase II) have been reported as clinical trials thereof (Melnikova I. Nat Rev Drug Discov 2007, 6, 863-864). Furthermore, it was reported that a delivery system of siRNAs in human cancer therapy is possible by using cyclodextrin-based nanoparticle polymers having transferrin as their target (CALAA-01; Calando Pharmaceuticals, Pasadena, Calif., USA; clinical phase I) (Oh Y K. et al., Adv Drug Deliver Rev 2009, 61, 850-862).
However, siRNAs are in vivo degraded within a short time due to their low stability and the anionic nature thereof hinders them from readily penetrating cell membranes with the same negative charge, leading to low transmissibility into cells, and thus there is a demand for a technology of preparing vehicles for efficient intracellular delivery. Accordingly, in order to efficiently deliver siRNAs into cells, there is needed an effective novel delivery system that has resistance against degradation enzymes, circulates in the living body for a long time, reaches target cells via a clinically available injection route, and enables an effective cytoplasm release after cell penetration.
As existing siRNA delivery vehicles, recombinant plasmids or viral vectors of expressing siRNA have been used, or lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticle, cationic polymer, or liposome-based delivery vehicles have been usually used. However, viral delivery vehicles are restricted by the size of a gene to be delivered and they do not guarantee in vivo stability thereof because they might cause immune side effects due to the immunogenicity of the surface proteins of the viral vectors. Further, the delivery vehicles using cationic molecules or synthetic polymers have showed low intracellular delivery efficiency and had cytotoxicity problems which might be caused during intracellular gene delivery procedures.
The vehicles using cationic molecules or cationic synthetic polymers that have been most frequently reported exhibit excellent delivery efficiency of plasmid DNA, but have a problem of low intracellular delivery efficiency of siRNA due to the low anionic charge density and intrinsic rigid structure of siRNA having a relatively short length of 20-23 mer, compared to plasmid DNA (Lee S H. et al., Acc. Chem. Res. 2012, 45, 1014-1025). That is, when a bond between siRNA and the cationic polymer vehicle forms by cation-anion electrostatic interactions, the low anionic charge density and intrinsic rigid structure of siRNA hinder compact structure formation of siRNA/vehicle complex, leading to formation of loose siRNA/vehicle complex. Thus, the bound siRNA could be easily attacked by degradation enzymes or negatively charged proteins in the blood stream.
Further, cationic synthetic polymer vehicles destroy cell membrane or mitochondrial membrane during intracellular delivery of siRNA due to high cationic charge density and limited biocompatibility, thereby generating a problem of cytotoxicity causing cell necrosis or cell death (Cho K C. et al., Macromol. Res. 2006, 14, 348-353). In order to solve this toxicity problem, many trials have been conducted to prepare various novel cationic polymers having reduced toxicity or to develop cationic polymer vehicles having reduced toxicity by modifying the existing cationic polymers. However, there is a still problem of low intracellular delivery efficiency due to the low charge density and intrinsic rigid structure of siRNA.